Regulation of MLK3 by oxidative stress in colon cancer cells

结肠癌细胞中氧化应激对 MLK3 的调节

• 批准号: 9097305
• 负责人: Deborah N Chadee
• 金额: $ 44.25万
• 依托单位: UNIVERSITY OF TOLEDO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9097305
• 关键词: Affect; Apoptosis; Binding; Breast Cancer Cell; Cell Death; Cell Proliferation; Colon Carcinoma; Colorectal Cancer; Feedback; Gene Expression; Goals; Growth; Human; Hydrogen Peroxide; In Vitro; Inflammation; Inflammatory; MAP Kinase Gene; MAPK Signaling Pathway Pathway; MAPK3 gene; MAPK8 gene; Maintenance; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of gastrointestinal tract; Malignant neoplasm of lung; Malignant neoplasm of ovary; Mediating; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Mitogens; Necrosis; Normal Cell; Oxidative Stress; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Physiological; Production; Proteins; Reactive Oxygen Species; Regulation; Role; Serine; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Stimulus; Stress; TNF gene; Threonine; Toxin; Tumor Cell Line; Ubiquitination; Up-Regulation; cancer cell; chemotherapeutic agent; cytokine; irradiation; malignant phenotype; metaplastic cell transformation; migration; mixed lineage kinase 3; novel; novel strategies; overexpression; protein degradation; public health relevance; research study; response; scaffold

 DESCRIPTION (provided by applicant): Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase (MAP3K) that regulates multiple MAPK signaling pathways in response to mitogenic and stress stimuli. Depending on the cellular context, MLK3 can phosphorylate MKK4 or MKK7 MAPK kinases (MAP2Ks), which activate c-Jun N-terminal Kinase (JNK) to trigger cell death. Alternatively, MLK3, through a kinase-independent scaffold function, can activate B-Raf and extracellular signal-regulated kinase (ERK) signaling to promote cell proliferation. MLK3 is required for the migration and invasion of human ovarian, gastrointestinal, lung, and breast cancer cells. A requirement for MLK3 in mitogen-dependent ERK activation and colon cancer cell proliferation has been demonstrated; however its function in colon cancer cell invasion is not known. Many cancer cells have persistent, intrinsic oxidative stress and higher levels of reactive oxygen species (ROS) than normal cells. Stimuli such as pro-inflammatory cytokines, irradiation, toxins and chemotherapeutic drugs can trigger ROS production. At very high levels ROS can elicit activation of signal transduction pathways and induce apoptosis or necrotic cell death, and at low levels ROS can stimulate cell proliferation. Our preliminary findings indicate that oxidative stress strongly activates ERK1/2 signaling which promotes the phosphorylation of MLK3 in colon cancer cells. Oxidative stress also promotes an ERK-dependent increase in MLK3 protein level and a decrease in MLK3 kinase activity. Furthermore, we demonstrated that ERK could phosphorylate MLK3 in vitro. Thus, we have identified ERK as a novel, positive regulator of MLK3 protein level and a negative regulator of MLK3 kinase activity in response to oxidative stress. We postulate that the enhancement of inactive MLK3 protein in response to oxidative stress could cause persistent MLK3-dependent stimulation of ERK signaling and thereby promote cellular transformation and proliferation of colon cancer cells. The overall goal of this proposal is to define the mechanism by which oxidative stress induces ERK-dependent regulation of MLK3, and elucidate the impact of this regulation on MLK3-dependent stimulation of cellular transformation, proliferation and apoptosis in colon cancer cells. Accordingly the specific aims of the proposal are: 1) To investigate the role of ERK signaling in H202-mediated upregulation of MLK3 in colon cancer cells. 2) To determine the impact of ROS and ERK activation on MLK3 kinase activity and function, 3) To elucidate the function of MLK3 in colon cancer cell proliferation and invasion. The experiments described in this proposal will allow us to gain a thorough understanding of how oxidative stress modulates MLK3 protein level and kinase activity and how this affects MLK3-dependent activation of MAPK signaling, proliferation and invasion of colon cancer cells. Furthermore, we will examine novel strategies to deplete cellular MLK3 protein levels to inhibit colon cancer cell proliferation and invasion.

 描述（由申请人提供）：混合谱系激酶3（MLK 3）是一种丝氨酸/苏氨酸促分裂原活化蛋白激酶（MAP 3 K），可调节多种MAPK信号通路，以响应促分裂和应激刺激。根据细胞环境，MLK 3可以磷酸化MKK 4或MKK 7 MAPK激酶（MAP 2Ks），其激活c-Jun N-末端激酶（JNK）以触发细胞死亡。或者，MLK 3通过激酶非依赖性支架功能，可以激活B-Raf和细胞外信号调节激酶（ERK）信号传导以促进细胞增殖。MLK 3是人卵巢癌、胃肠道癌、肺癌和乳腺癌细胞迁移和侵袭所必需的。已证实MLK 3在促分裂原依赖性ERK活化和结肠癌细胞增殖中的需要;然而，其在结肠癌细胞侵袭中的功能尚不清楚。许多癌细胞具有持续的内在氧化应激和比正常细胞更高水平的活性氧（ROS）。促炎细胞因子、辐射、毒素和化疗药物等刺激物可触发ROS产生。在非常高的水平ROS可以引发信号转导途径的激活，并诱导细胞凋亡或坏死性细胞死亡，在低水平ROS可以刺激细胞增殖。我们的初步研究结果表明，氧化应激强烈激活ERK 1/2信号，促进结肠癌细胞中MLK 3的磷酸化。氧化应激还促进MLK 3蛋白水平的ERK依赖性增加和MLK 3激酶活性的降低。此外，我们证明ERK可以在体外磷酸化MLK 3。因此，我们已经确定ERK作为一种新的，积极的MLK 3蛋白水平和MLK 3激酶活性的负调节剂，在氧化应激反应。我们推测，无活性MLK 3蛋白在氧化应激反应中的增强可能导致持续的MLK 3依赖性ERK信号刺激，从而促进结肠癌细胞的细胞转化和增殖。该提案的总体目标是确定氧化应激诱导MLK 3的ERK依赖性调节的机制，并阐明这种调节对结肠癌细胞中MLK 3依赖性刺激细胞转化、增殖和凋亡的影响。因此，该提议的具体目的是：1）研究ERK信号传导在结肠癌细胞中H2 O2介导的MLK 3上调中的作用。2)探讨ROS和ERK激活对MLK 3激酶活性和功能的影响。3）阐明MLK 3在结肠癌细胞增殖和侵袭中的作用。本提案中描述的实验将使我们能够全面了解氧化应激如何调节MLK 3蛋白水平和激酶活性，以及这如何影响MLK 3依赖的MAPK信号转导激活，增殖和结肠癌细胞的侵袭。此外，我们将研究新的策略来消耗细胞MLK 3蛋白水平，以抑制结肠癌细胞的增殖和侵袭。